Mullerian inhibiting substance (MIS), a member of the TGF-beta family of growth and differentiation factors, has the essential role during male embryonic sexual differentiation of causing involution of the Mullerian ducts, the anlagen of the female internal reproductive tracts. The unexpected phenotypes of Leydig cell hyperplasia and neoplasia in mice with targeted deletions of the genes for MIS or its receptor, however, raised the prospect of a novel role for MIS in the developing testis. These data suggested that MIS has an essential role in helping to maintain a normal complement of Sertoli and Leydig cells in the maturing testis. This proposal will test the hypothesis that MIS prevents the unrestrained proliferation of immature Leydig cells and modulates their responsiveness to mitogenic factors in the maturing testis. The Specific Aims of this proposal are to: 1.) Characterize the expression of the MIS type II receptor in Sertoli and Leydig cells to verify the ability of MIS to signal in these cells, and examine the anti-proliferative role of MIS in primary Leydig and Sertoli cells to help elucidate the interplay of hormonal influences on the control of cellular proliferation in the developing testis. 2.) Delineate the testicular phenotypes (cell numbers and differentiated function) of MIS transgenic and knockout mice by stereologic studies and in vivo assessment of proliferation, and examine the anti-proliferative activity of MIS in these animal models, and 3.) Investigate the molecular mechanisms by which MIS inhibits Leydig cell proliferation by determining whether MIS causes cell cycle arrest or apoptotic cell death. These studies to elucidate the paracrine and autocrine actions of MIS in developing Leydig and Sertoli cells will advance our understanding of a novel pathway for control of cellular proliferation in the postnatal testis. Inhibitors of proliferation are increasingly being recognized as having critical roles in counter-acting the actions of mitogenic agents to achieve balanced tissue growth and morphogenesis. Thus the anti-proliferative action of MIS on the testicular somatic cells may help control testicular morphogenesis and function; processes that are essential for normal sexual differentiation and attainment of secondary sexual maturation and reproductive fertility. Moreover, clarification of the pathway by which MIS prevents unregulated cellular proliferation and neoplastic transformation of Leydig cells will provide insights into the pathogenesis of testicular tumorigenesis.